Project Summary In this Phase I STTR project, Photonic Pharma LLC (PP), in collaboration with the University of Minnesota (UMN), will establish a technology platform for high-throughput screening (HTS) to discover small-molecule inhibitors of pathological self-association of tau and ?-synuclein (?syn). Oligomers of these proteins are implicated in the pathology, severity, and progression of Alzheimer?s disease (AD) and Alzheimer?s disease related dementia (ADRD). This is novel and distinct from previous strategies that have unsuccessfully targeted fibrillar tau/?syn. Thus, compounds that selectively target tau-tau and ?syn-?syn interactions within toxic oligomers are urgently needed. PP?s proprietary combination of fluorescent biosensor engineering and fluorescence lifetime (FLT) detection provides the capability to detect, with precision 30 times greater than conventional HTS technology, small changes in protein structure and interactions in cells and cell-free systems. This precision is essential for reliable detection of structural changes within these toxic oligomers. Aim 1 will optimize our tau and ?syn live-cell and cell-free biosensors, scaling up production and seeking the sensitivity and reproducibility needed to perform primary HTS on a 50k-compound CNS-focused chemical collection. Multiple orthogonal approaches are taken to ensure targeting of oligomers as opposed to fibrils. Our biosensors have shown effectiveness and robustness in pilot HTS with small libraries of known biologically active compounds (Z? > 0.7 for both sensors, indicating high assay quality). From these pilot screens, we have identified Hits with saturable FRET profiles, functional efficacy, and direct target engagement. These Hits will serve as tool compounds for FRET assay optimization as we scale up for the 50k-compound HTS. Following those screens, Hits (molecules that attenuate FRET by ? 5SD) will undergo concentration- response rescreening to determine their apparent potency (EC50) for the modulation of tau-tau or ?syn-?syn interactions, seeking compounds with sub-micromolar affinity and saturable response. Aim 2 will apply secondary assays to determine the functional relevance and mechanism of action (MOA) of Hits identified in Aim 1. The most potent Hits will be prioritized for functional analysis in an overexpression model of tau/?syn induced cytotoxicity. These functional Hits will then be parsed using cell-free biosensors to identify potential direct (on-target) versus indirect MOA. Hits with direct MOAs will be further characterized with orthogonal biochemical, biophysical and functional assays. This Phase I project will validate a large-scale HTS platform and initiate the Hit-to-Lead process for Hits that interact with tau or ?syn. A subsequent Phase II project will evaluate in detail MOA and structure-activity relationships and initiate pre-clinical animal studies for the most potent compounds. We have formed collaborations with experts in AD and ADRD and with executives in the pharmaceutical industry, to ensure that the lead compounds discovered in this project will be further developed for therapeutic discovery.